The identification and localization of biomolecules, throughout entire brain circuits, with nanoscale precision would enable many fundamental insights into the mechanisms underlying the operation of normal and pathological neural networks. We recently discovered that we could physically magnify specimens by embedding them in a dense swellable polymer, anchoring key biomolecules to the polymer mesh, and adding water to swell the polymer, a process we call ‘expansion microscopy’1. Despite the high isotropy of the expansion process, the initial polymer recipe enabled just 4-4.5x expansion, or roughly 60-70 nm spatial resolution. Ideally it would be possible to improve the expansion chemistry so as to enable, ultimately, the imaging of membrane boundaries, as well as protein complexes. Here, we report on an iterative ExM (iExM) chemistry that can achieve ~20x physical magnification of mouse brain tissues, or 20-nm lateral resolution on conventional optical microscopes2. As with the first version of ExM, iExM-processed samples are optically clear. Thus, iExM may be useful for imaging nanoscale neuronal structures such as synaptic clefts over entire neural circuits in intact mammalian tissues. Brain circuit mapping using iterative ExM may open up a variety of insights into the underpinnings of behavior, cognition, and disease.